JP3812949B2 - Hepatitis B vaccine - Google Patents

Abstract

PCT No. PCT/GB95/00208 Sec. 371 Date Dec. 28, 1995 Sec. 102(e) Date Dec. 28, 1995 PCT Filed Feb. 2, 1995 PCT Pub. No. WO95/21189 PCT Pub. Date Aug. 10, 1995A variant or so-called "escape mutant" HBsAg protein or fragment thereof displaying the antigenicity of hepatitis B virus surface antigen is disclosed, in which the mutant protein or fragment thereof (mHBsAg) comprises a modified 'a' determinant in which at least two amino acids are inserted downstream of position 122 of the wild type HBsAg sequence. A vaccine comprising the mHBsAg is provided, as is a kit for diagnostic in vitro detection of anti-mHBsAg antibodies and an antibody preparation comprising anti-mHBsAg antibodies.

Description

The present invention relates to a recombinant DNA molecule encoding a polypeptide having specificity for hepatitis B virus antigen.
More particularly, the present invention relates to a vaccine composition that stimulates the production of human antibodies against mutant hepatitis B virus.
Cloning of the genomes of various serotypes of hepatitis B virions is known (Miller et al.). Dane particles isolated from infected patients who are hepatitis B virions have a diameter of about 42 nm. Each is composed of hepatitis B surface antigen (HBsAg), capsid (HBcAg), endogenous polymerase and DNA genome. A third polypeptide, “e” antigen (HBeAg), is made by hepatitis B virus and is found solubilized in serum.
Although infection with hepatitis B virus (HBV) has become a serious problem worldwide, vaccines used for mass immunization are now widely used. Commercially available vaccines against HBV consist of hepatitis B virus surface antigen (HBsAg) having either natural or recombinant forms. The canonical (or wild type) hepatitis B surface antigen can be recovered from the plasma of a patient infected as approximately 22 nm particles consisting of two proteins known as P24 and its glycosylated derivative GP28, and these The two proteins are both encoded by the S protein coding sequence or the 226 amino acid coding sequence on the HBV genome known as the HBV S-gene (Tiollais et al. (1985)). The complete amino acid and nucleotide sequence encoding HBsAg is described in Valenzuela et al., Nature, 280, page 815 (1979). The numbering system used by Tiollais et al. To identify nucleotide and amino acid positions is used herein.
Inserting the coding sequence of the HBV S-gene under the control of a yeast promoter onto an expression vector allowing expression of HBsAg in S. cerevisiae for the production of vaccines Harford et al. In Development Biol. Standard, 54, p. 125 (1983), Valenzuela et al. In Nature, 298, p. 347 (1982) and bitter ( Bitter) et al., J. Med. Virol., 25, p. 123 (1988). Expression in Pichia pastoris has been reported by Gregg et al., Biotechnology, Volume 5, page 479 (1987) (see also European Patent Publication No. 0226846). Expression in Hansenula polymorpha has also been reported (European Publication No. 0299108).
The vaccine was also prepared from hybrid immunogenic particles consisting of HBsAg protein as described in European Patent Publication No. 0279940. Such immunogenic particles include, for example, all of the HBsAgpr precursor protein encoded by a coding sequence that rapidly advances the HBV-S gene on the HBV genome, referred to herein as the Pre-S coding sequence. Or some can be included. This Pre-S coding sequence generally consists of 163 amino acids ( ay HBV subtype) and consists of a Pre-S1 coding sequence and a Pre-S2 coding sequence. The latter encodes 55 amino acids and rapidly advances the coding sequence for the S protein. (European publication number EP-A-0278940).
The surface reading (S antigen) open reading frame of HBV-DNA is divided into three regions, Pre-S1, Pre-S2 and S. This open reading frame encodes three envelope proteins of HBV termed large, middle and major proteins. The major protein, HBsAg, is composed of 226 amino acids and is encoded by the S gene (Tiollais et al. (1981); Tiollais et al. (1987) and Lau et al.). All three envelope proteins contain the antigenic site of HBsAg and are easily detected by known immunoassays for HBsAg. These immunoassays are widely used to diagnose HBV infection and to widely screen blood donors. The reactivity of HBsAg depends on the structural conformation of the hydrophilic region from amino acids 124-147 defined as the “a” determinant (Ashton-Rickardt et al. And Brown) Et.) These are common to all HBV subtypes, and antibodies against them confer protection against reinfection by either subtype.
HBV-DNA sequences have been shown to hybridize with HBV probes under very harsh conditions in liver, serum and blood mononuclear cells of patients negative for serum HBsAg (Brechot et al. (1985). Year) and Thier et al.). Recent results from various experiments using dot blot hybridisation or polymerase chain reaction (PCR) confirm the presence of HBV DNA sequences in the sera of patients negative for HBsAg. The development of PCR technology enables detection of very low levels of HBV replication in many patients and the analysis of the sequence of many isolates, which allows genetic diversity for some virus isolates. (Carman et al. (1990); Brechot et al. (1991); Blum et al.). In Taiwan and the Kingdom of Sardinia, where HBV is quite endemic, 1.7% and 0.3% of HBsAg-negative healthy blood donors possess HBV DNA in serum that can be detected by dot blot hybridization. (Lai et al. (1989) and Sun et al.). In the mainland of China, molecular epidemiological studies using PCR in anti-HB-positive patients have HBV carriers in which HBsAg is undetectable, and such people account for 3% of the general population in China. (Luo et al.).
The HBV antigen subtype was serologically defined and was shown to be caused by a single base change in the genomic region encoding HBV (Okamoto et al.). However, all currently known antigen subtypes contain an “a” determinant composed of amino acids 124-147 of HBsAg. Antibodies against the “a” determinant confer protection to all subtypes. In vitro mutagenesis has shown that the cysteine at position 147 and the proline at position 142 are important for the total antigenic expression of the “a” determinant (Ashton-Rickardt et al.).
In addition, Howard Thomas and William Carman reported in WO 91/14703 a variant HBsAg fragment in a vaccinated child born from a mother infected with HBV. Sequencing revealed that a point mutation from guanosine to adenosine at the 587th nucleotide position resulted in an amino acid change from glycine to arginine at position 145 in the “a” determinant of HBsAg (Kerman (See also (Carman) et al. (1990)). Similar HBV mutants have been reported to replicate in the host under humoral immune pressure, activated (vaccine) or passively (hyperimmunoglobulin) induced. (Okamoto et al. (1992) and McMahon et al.). However, the long-term presence of HBsAg and anti-HBs (vaccine induction) is detected by known immunoassays in some of the patients' sera, suggesting that the antigenicity is not completely lost by mutation (Carman et al. (1990), Okamoto et al. (1992) and McMahon et al.).
From a clinical and epidemiological point of view, it is more important to investigate the molecular form of a patient's HBV without causing HBsAg response in a standard assay. From the results of sequencing from Japanese patients who are HBeAg and HBV-DNA and anti-HBs positive, the 9th to 22nd amino acids of the Pre-S2 region were deleted, and the 3rd and 8th positions of Pre-S2 , And the amino acid at position 126, 131 and 133 of the first loop of the “a” determinant of HBs was shown to be substituted (Moriyama et al.). Deduced amino acid sequence analysis from other isolates reveals substitutions at positions 3, 53, and 210 of the major HBs protein, all of which are outside the common “a” determinant, and serum The disappearance of HBsAg in it cannot be explained (Liang et al.).
During the last decade, various putative variants or mutants of hepatitis B virus have been described. For example, McMahon et al. Described a glycine to arginine substitution in the monoclonal antibody binding domain on the putative HBsAg (estimated by DNA sequence analysis) in liver transplant patients treated with anti-HBsAg monoclonal antibody. (Cold Spring Harbor Symposium on the Molecular Biology of Hepatitis B Viruses, September 1989).
A modified “a” determinant in which two specific amino acids, asparagine and threonine are inserted at position 122 of the HBsAg sequence in the investigation by Carman et al. And in the subject of patent application WO 94/26904. A mutant hepatitis B virus is described. In addition, the mutation in which the wild-type arginine is replaced with glycine at position 145 is also described in detail in the list of amino acid sequences.
According to other reports, children and adults with circulating hepatitis B surface antigen have been found and are special after immunization with one of two approved hepatitis B vaccines. The virus is shown to replicate despite the presence of other antibodies (anti-HBs) (Zanetti et al.). Analysis of HBsAg using monoclonal antibodies indicated that circulating antigens do not have an “a” determinant or that the determinant is masked. It was concluded that the emergence of a variant of hepatitis B virus was detected, probably due to epidemiological pressures associated with immunization in areas where the infection is a local disease. However, further characterization of this variant was not revealed.
From the work of Zanetti et al., At least in susceptible hosts with immunogenic properties, it produces `` escape mutants '', i.e., replicating infectious viruses that are mutated to not neutralize immunity. Is clearly a major drawback of currently used hepatitis B vaccines. Such a mutant virus clearly has the ability to cause disease and is believed to be inherited. Therefore, this mutant virus creates significant immunization problems because it is not effectively neutralized by antibodies produced by normal HBsAg-based vaccines. Other mutations have also been reported in HBV, but the importance regarding these altered antigenicities is unclear (Moriyama et al. And Lai et al. (1990)).
In China, where HBV infection is a very local disease, recent research using PCR has resulted in either idiopathic cirrhosis, active chronic hepatitis (CAH) or persistent chronic hepatitis (CPH). It is suggested that 30-40% of all HBsAg negative patients have HBV-DNA replicating in serum or liver tissue (Zhang et al.). These studies indicate that HBV variants are present in the Chinese population and have the property that HBsAg is undetectable in serum. Surprisingly, such putative mutants have not been characterized at the molecular level.
Isolates from Chinese patients (ie patients with no detectable HBsAg) are insertion mutants with additional amino acids inserted between the 122 and 124 codons immediately before the “a” determinant ( insertion mutant). Such sequence alterations have not been previously reported for known HBV subtypes and were not found in 30 HBsAg positive Chinese patients from the same region. Interestingly, the region where the insertion occurred is an important epitope region of HBsAg. The inserted sequence is known to be immediately downstream of the codon at position 122 that determines the subtype determinant (d) and immediately upstream of the “a” determinant. The nucleotide and amino acid sequences before and after the codon at position 122 are all the same as those described for wild-type HBV (FIGS. 6 and 7). While not wishing to be bound by theory, we believe that such mutations result in conformational changes that affect epitopes of the “a” determinant and “d” subtype. One possibility is that the epitope can be changed by affecting the spatial organization of the two loops of the “a” determinant by insertion of an amino acid. Another possibility is that the first loop of the “a” determinant, defined by a monoclonal antibody (Waters et al.), Contains an amino acid upstream (rather than previously thought). It is done. We have shown that the above amino acids are formed by disulfide bridges upstream of 122 rather than 124 as previously suggested (Waters et al. And Ashton-Rickardt et al.). Report. The inserted amino acid increases the distance of the loop from 14 amino acids to 16 or 17 amino acids, thereby changing the conformation of this loop and inhibiting neutralizing antibody binding.
In the present invention, we have isolated HBV isolated from Chinese patients whose sera are positive for HBV-DNA by dot blot hybridization but are HBsAg negative in a commercially available polyclonal antibody-based immunological determination. Report a new variant or “escape mutant”. Furthermore, the present invention overcomes or at least alleviates the drawbacks associated with these vaccines, since known HBV vaccines are not effective with the newly discovered mutants described above.
The present invention provides the properties of a newly identified mutant of HBV in which at least two amino acids have been inserted immediately downstream of position 122 of the HBV envelope region. The present invention also provides methods for determining the presence of mutant HBV in a test sample and reagents that can be used in these methods. All the concepts of the present invention have an insertion of at least 2 amino acids downstream of position 122 of the HBsAg sequence, corresponding to the insertion of at least 6 nucleotides downstream of the 519th nucleotide of the HBsAg genome. Also provided are modifications of the “a” determinant.
Nucleic acid sequences derived from mutated HBV, or portions thereof, can be used as probes to determine the presence of mutated HBV in a test sample. This sequence can also utilize a polypeptide sequence of a mutant HBV antigen encoded within the genome of such a mutant HBV and is useful as a standard or reagent in a diagnostic test and / or as a vaccine component Enables the production of Monoclonal and polyclonal antibodies against epitopes contained within the polypeptide sequence are also used for diagnostic tests as well as therapeutic agents, for screening antiviral agents, and for single mutant HBV from which the nucleic acid sequence is derived. Can be used for separation purposes.
It is understood that the mutant HBsAg may contain a Pre-S sequence if necessary.
The mutant HBsAg protein or fragment thereof according to the present invention is hereinafter abbreviated as “mHBsAg”.
These mHBsAg mutants are preferably synthetic or highly purified material that is not in “naturally occurring” form and free of blood products.
Preferably, these mutants described in the present invention correspond to full length HBsAg and are the same as wild type HBsAg except that at least two amino acid residues are inserted downstream of position 122. is there.
Preferably, the HBsAg mutant has a highly purified form, eg, 75% or more purity, more preferably 90% or more purity, and most preferably 95-100% purity.
According to a further concept of the present invention, there is provided a vaccine composition comprising an immunoprotective amount of the above “escape mutant” of HBsAg combined with a suitable carrier.
Other concepts of the present invention are described below.
The mHBsAg and vaccines of the present invention are used to solve the problems considered by the emergence of “escape mutants” as described above in which the region immediately upstream of the “a” determinant of the viral HBsAg (envelope region) has been modified. Is done. In particular, the vaccine of the present invention is protected against a variant HBV defined as having an HBV envelope region modified in the HBsAg amino acid sequence with at least two amino acids inserted downstream of position 122. Or has the advantage of being used to prevent the occurrence of inheritance of such mutant forms of HBV.
Accordingly, there is also provided a method for protecting humans against disease symptoms associated with infection with mutant HBV comprising administering a safe and effective amount of a vaccine according to the present invention to humans.
According to another concept of the invention, mHBsAg intended to be used for treatment, in particular prevention, is provided.
The present invention also provides the use of mHBsAg in the manufacture of a vaccine composition intended to protect humans against disease symptoms associated with infection with mutant HBV.
When used to immunize a human against an existing mutant HBV virus, the mHBsAg sequence in the vaccine generally matches or antigenically matches the mHBsAg sequence in the mutant HBV virus. It is considered equivalent.
Preferably, the amino acid residue inserted after position 122 in the mHBsAg of the present invention is derived by insertion of 6 or more nucleotides. The inserted nucleotide encoding the extra amino acid affects any of the three nucleotides at the 123rd codon in normal HBsAg.
Theoretically, mutations that change the hydrophilicity of the envelope protein can change the antigenicity and allow mutant HBV to appear. The hydrophilicity of the wild type and that of the mutated region are completely different. The loss of hydrophilicity described herein is often correlated with a loss of antigenicity. Increasing the hydrophilicity of the mutant in the first loop results in a converted protein of the major protein in the envelope lipid, which results in a significant conformational effect.
In a preferred embodiment of the invention, the residue after position 122 of normal HBsAg reduces the hydrophilicity of the “a” determinant.
According to another preferred embodiment of the invention, mHBsAg is the same as the normal (wild-type) HBsAg S-protein except that at least two amino acid residues are inserted downstream of position 122. .
According to a particularly preferred embodiment of the invention, the modification downstream of position 122 is to insert arginine and then alanine, respectively. It is also preferable to insert arginine, glycine and alanine downstream of the 122nd position in this order.
Preferred embodiments of the concepts of the present invention are the same as the other concepts, with modifications as necessary.
The invention is illustrated in detail by the following examples. This example refers to the following drawings:
FIG. 1 shows the aminotransferase (ALT) level in the continuous serum of patient # 1. Normal levels are less than 40 μi / liter. Serum HBV markers were measured at various indicated times. In August 1992, HBV DNA was detected by PCR, but HBsAg and other HBV markers were not detected.
FIG. 2 shows the continuous serum ALT level and hepatitis B virus marker in patient # 2. Abbreviations: w indicates wild type; m indicates mutant.
FIG. 3 graphically illustrates the surface gene open reading frame, the location of PCR and sequencing primers, and the overlapping polymerase open reading frame (P ORF). The shaded circles indicate start and stop codons, and the shaded squares indicate insertion hot spots (positions 522-593) for the “a” determinant. Nucleotide numbering is from a temporary EcoRI site, as is the known HBV DNA sequence, since the isolate does not have an EcoRI site.
FIG. 4 shows the nucleotide inserts (left and right) of isolates 1 and 2 after direct sequencing of the PCR product compared to the wild type (middle). Arrows indicate insertion points.
FIG. 5 shows the nucleotide and amino acid sequences of isolates 1 and 2 (left and right) as wild type ( adw ) Is shown. The inserted sequence is underlined.
FIG. 6 shows the complete nucleotide sequence of mHBsAg isolate 1. The upper sequence represents the surface gene and “a” determinant sequence of the mutant HBsAg isolate, and the lower sequence represents the wild type sequence. (---) indicates a point where an amino acid was inserted.
FIG. 7 shows the complete nucleotide sequence of mHBsAg isolate 1. The upper sequence represents the surface gene and “a” determinant sequence of the mutant HBsAg isolate, and the lower sequence represents the wild type sequence. (---) indicates a point where an amino acid was inserted.
Table 1 shows the synthetic oligonucleotide primers used in the polymerase chain reaction and sequencing reaction.
Table 2 shows the positive / negative binding rates of serum HBsAg from patient numbers 1 and 2 to monoclonal antibodies, adw Wild type was used as a control.
Table 3 shows previously known wild type sequences ( adw ) And the nucleotide and amino acid sequence homologies of the mutant S gene compared to Chinese isolates from the same region as the wild type.
HBV mutants are more commonly found in specific regions of the sequence characterized by run of bases. In this case, studies have associated the insertion with four adenosine runs. This region is considered to be a “hot spot” for insertion in some isolates from Chinese HBsAg negative HBV carriers.
According to a further concept of the present invention, a method for preparing mHBsAg and a vaccine composition obtained therefrom are provided.
Preferably, mHBsAg is obtained synthetically by peptide synthesis or more preferably by recombinant DNA technology.
Methods for construction, manipulation and confirmation of recombinant DNA molecules and sequences are known in the art. In order to modify the HBV S protein of HBsAg to obtain the mHBsAg of the present invention, a codon CGG and GCA or other triplet codon combination encoding arginine and alanine, respectively, is inserted downstream of position 122. It is desirable. Alternatively, the codon CAC, GGG or GCG, or other triplet codon combinations encoding arginine, glycine and alanine, respectively, may be inserted between the nucleotides at positions 122 and 124.
Various methods can be used to modify the sequence appropriately. One preferred method is complete de novo synthesis of the desired coding sequence using viral or yeast codon frequencies by phosphite or phosphoramidite chemistry.
DNA synthesis is commercially available from various companies. Examples of such gene synthesis are described in Hayden and Mandecki, DNA, Vol. 7, page 571 (1988) and the like. As described in the cited reference.
The second method is suitable restriction from vectors already carrying the HBV genome, as described in Botstein and Shortle, Science, 229, p. 1193 (1982). The fragment is cloned onto a single stranded vector followed by site-specific in vitro mutagenesis. A culture of E. coli K12 C600 carrying the recombinant plasmid pRIT10601, in which the HBV genome of the ay subtype was cloned on pBR322, was given an American type under accession number ATCC 39132 on June 2, 1982. Deposited to the Culture Collection (American Type Culture Collection) based on the Budapest Treaty. Sequences encoding the S-gene specifying the 226 amino acid HBsAg protein or longer sequences encoding the Pre polypeptide can be excised from such clones by standard recombinant DNA techniques.
A suitable restriction fragment is the 575 bp XbaI-AccI fragment within the S-gene coding region of pRIT10601. Vector systems used for in vitro mutagenesis are commercially available. The mutated gene fragment thus obtained is reinserted into the S-gene.
The third method involves the targeted mutation using polymerase chain reaction (PCR) technology as described in Ho et al., Gene, 77, p. 51 (1989). Make changes.
In each case, the mHBsAg coding sequence is expressed in a suitable host under the control of a suitable promoter.
Expression vectors having a DNA sequence encoding mHBsAg are novel and form a further concept of the present invention. Hosts transformed with the above expression vectors also form yet another concept of the present invention.
According to a preferred concept, Saccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha is used as the host and the expression is expressed in the yeast TDH3 promoter (glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde). -3-phosphate dehydrogenase gene, Valenzuela et al., 1982; see Bitter et al., 1988) or PH05 (Miyanohara et al., 1983), MOX, FMDH (EP-A-0299108) No.) and AOX (see EP-A-0226846) and the like.
The transformed host can be cultured or fermented by known means to extract and purify mHBsAg. Purification of HBsAg from yeast cells is known in the art. S. No. 4,649,192; 4,683,294; 4,694,074 or 4,738,926. Purification of mHBsAg of the present invention is performed in a similar manner.
Vaccines containing mHBsAg are prepared by known techniques, preferably contain an immunoprotective amount of mHBsAg in buffered saline, and include various known adjuvants such as aluminum hydroxide and aluminum phosphate. Mix or adsorb with either. “Immunoprotective” means that a sufficient amount of mHBsAg induces an immune response mediated by sufficient protective antibodies or cells and provides protection against infectious agents without serious side effects Means to be administered. The dosage of mHBsAg varies depending on whether the vaccine is adjuvanted, but is usually in the range of 1-1000 μg protein. Preferably 1 to 200 μg of protein is used, more preferably 5 to 40 μg of protein. The dose and the number of doses are determined within a standard dose range in consideration of the antibody titer of the patient and other responsiveness.
mHBsAg may also be mixed with normal HBsAg or other HBsAg such as homogeneous or complex HBsAg particles that contain all or part or parts of PreS1 or PreS2 polypeptide for vaccine formulation. In addition, mHBsAg may be mixed with hybrid HBsAg particles having epitopes derived from proteins of other organisms and with other immunogens to form bivalent or multivalent vaccines. Vaccine preparations are usually described in “Vaccines” (Voller et al., University Park Press, Baltimore, MD, USA, 1978).
mHBsAg can be used for inclusion as an immunoreagent in a detection kit for mutated HBV virus infection. In addition, mHBsAg may be used to obtain a polyclonal or monoclonal antibody by a known method. In this case, the monoclonal antibody may be specific to a mutant antigen and not recognize normal HBsAg. .
According to other embodiments of the present invention, the antibodies that immunologically react with sera containing mutant HBV antibodies and complexes thereof, and against epitopes specific for mutant HBV in these polypeptides The antibodies can be used in immunoassays to detect the presence of mutant HBV antibodies, or the presence of viruses and / or viral antigens in biological test samples. The design of these immunoassays has changed and such changes are known in the art; these changes are described herein. The immunoassay is derived from a mutated HBV nucleic acid sequence comprising clones, from a mutated HBV nucleic acid sequence in these clones, or from a mutated HBV genome from which the nucleic acid sequences of these clones are derived. One viral antigen such as a polypeptide of the above may be used. Alternatively, the immunoassay may use a combination of viral antigens derived from the above sources. Furthermore, the immunoassay may use, for example, monoclonal antibodies against the same viral antigen, or polyclonal antibodies against various viral antigens. Assays include, but are not limited to, those based on competition, direct reaction or sandwich type assays. The assay may be one that uses a solid phase or may be performed by immunoprecipitation or other methods that do not use a solid phase. Assays using labels as signal generating compounds and examples of these labels are described herein. The signal is also an ongoing U.D. S. Amplified by using biotin or avidin, enzyme labeling or biotin anti-biotin system as described in patent application numbers 608,849; 070,647; 418,981; and 687,785 It may be. Recombinant polypeptides comprising epitopes derived from immunodominant regions of mutant HBV may be used for detection of viral antibodies in biological test samples of infected patients. It is also contemplated that antibodies may be used to distinguish between acute and non-acute infections. A kit that can be applied to immunodiagnostics and that contains appropriate reagents is suitable for conducting appropriate materials, as well as assays, such as mutant HBV epitopes or polypeptides of the invention containing antibodies against mutant HBV epitopes in appropriate containers. It is constructed by filling the required remaining reagents and materials, along with appropriate assay instructions.
Thus, according to a preferred concept of the present invention, the aim is in vitro detection for the diagnosis of anti-mHBsAg antibodies in biological media and in specific neutralizing antibodies after vaccine injection, characterized in that it consists of: The following kit is provided:
a) mHBsAg as defined herein; and
b) Means used to detect antigen-antibody reaction.
According to a further concept of the invention, there is provided an antibody preparation comprising anti-mHBsAg antibodies intended for use in the diagnosis, prevention or treatment of hepatitis B infection in humans. The present invention also provides a method for treating humans with an effective amount of the anti-mHBsAg antibody for preventing or treating hepatitis B infection.
The invention is illustrated in detail by the following examples.
Example
Patient number 1 was a 58 year old male with a 6 year history of non-A non-B chronic hepatitis. In August 1992, HBV-DNA was discovered by PCR in the absence of HBsAg and other HBV markers (FIG. 1). At this stage, the patient had cirrhosis.
Patient No. 2 is a 23-year-old woman from the south of China. In a routine test in March 1993, serum aminotransferase (ALT) levels were slightly elevated, and HBsAg and HBeAg were positive. Although both immunoglobulin (Ig) M and IgG anti-HBs were negative. In a follow-up in January 1994, she was still positive for HBV-DNA and positive for anti-HB, but negative for HBsAg, HBeAg, and anti-HBs (FIG. 2).
Two patients were negative for hepatitis C virus (HCV) and HCV-RNA. We studied 30 HBsAg positive Chinese patients from the same region who suffer from chronic liver disease or hepatocellular carcinoma.
Serological investigation
HBsAg, HBeAg, total anti-HBc, IgM anti-HBs and anti-HBs were tested by commercially available enzyme immunoassays (Abbott Laboratories, North Chicago, Illinois, USA). HBsAg and anti-HBs results were confirmed in the UK.
Monoclonal antibody binding
Known to bind to the “a” determinant—panel anti-HBs monoclonal antibodies were used to define more specific changes in the epitope of the “a” determinant. Polystyrene beads (Northumbria Biologicals, Northumberland, UK) were coated with antibodies RFHBs-1, RFHBs-2 and RFHBs-7, followed by phosphate buffer (pH 7.2). Incubated with serum samples diluted 1: 2 with 50% newborn calf serum. Bound HBsAg was obtained from the “AusRIA II” kit (Abbott Diagnostics). ¹²⁵ When detected using I-anti-HBs, RFHBs-1 and RFHBs-2 bind to a cyclic peptide containing the 124th to 137th amino acid sequence and RFHBs-7 to a cyclic peptide derived from the 139th to 147th amino acid. Combined.
Hepatitis marker
HBsAg, HBeAg, total anti-HBc, IgM anti-HBc, and anti-HBs were tested by enzyme immunoassay (Abbott Laboratories, North Chicago, Ill.).
Dot blot hybridization
HBV-DNA was detected by dot blot hybridization modified by the method described in Welller et al., J. Med. Virol., 9, pp. 273-280 (1982).
HBV-DNA extraction, PCR and direct sequencing
50 μl serum for 2 hours at 68 ° C. in a volume of 200 μl 0.5% sodium dodecyl sulfate, 25 mM sodium acetate, 2.5 mM EDTA, 1 mg / ml proteinase k (Boehringer Mannheim, Lewes, UK Digestion in the presence of the country, extraction with phenol was performed twice, extraction with chloroform was performed twice, and HBV-DNA was further precipitated with ethanol. After washing twice with 70% ethanol, the DNA pellet was resuspended in 10 μl water.
5 μl of extracted HBV-DNA was used as a template for PCR amplification. 1 set of primers ( adw By using PO5 ′ (5′-TGCGGGTCACCATAT, position 2818-2833) and POL3 (5′-AAGGATCCCAGTGGC, position 1409-1395) (sub-type) as subtypes) (Table 2), Pre-S1, Pre -A 1800 bp fragment containing S2 and S genes was obtained (Figure 3). Another set of primers, M3 (5'-CTGGGAGGTGTGGGGGAGGAGAATT, positions 1781-1755) and 3C (5'-CTAACATTGAGATTCCCCGAGA, positions 2460-2439) were used to amplify the Pre-C and C regions. It was. Similar serum samples were analyzed separately in China and the United Kingdom using various primer sets.
Direct sequencing of PCR products was performed using the “fmol” Sequencing Kit (Promega Co., Southampton, England). The ends of the sequencing primers (Table 1) were added at 25 μCi in a volume of 10 μl using 10 units of polynucleotide kinase (Boehringer Mannheim) for 10 minutes at 37 ° C. ³² Labeled with P-adenosine triphosphate (Amerhsam International, Amerhsam, UK). 1.5 μl of the reaction mixture was used directly in the sequencing reaction. 90 μl of the PCR product was precipitated with an equal volume of 4 mol / liter sodium acetate and 2 volumes of isopropanol at room temperature for 10 minutes, pelleted by centrifugation for 10 minutes, and then washed twice with 70% ethanol. After resuspending in 20 μl of water, 9.5 μl of DNA was added to the reaction buffer containing end-labeled primers and 5 units of Taq enzyme (Promega Co.). Dideoxynucleotide termination sequencing was performed according to the manufacturer's instructions. This sequencing cycle was performed 30 times. The denaturation, annealing and expansion stages were 30 seconds, 30 seconds and 1 minute, respectively.
Cloning and sequencing of the S gene
Direct sequencing results showed that patient 1's first serum (February 1993) had a form of a mixture of wild type and mutant viruses. The serum sample PCR product and the PCR product after patient number 2 were cloned into the pUC19 vector and the results of direct sequencing were confirmed. The nucleotide sequence of the amplified HBV-DNA was determined using a sequenase DNA sequencing kit (version 2.0, USB, Cambridge, England).
Hydrophobic plot
The amino acids 122-137 and 122-139 or 140 (including the inserted amino acid) of the “a” determinant of HBsAg from wild type and mutant virus isolates, respectively, were inserted into Prosis software. (Pharmacia, St. Albans, England).
Dot blot hybridization
HBV-DNA could be detected in the serum of patient # 1 after two additional PCR amplifications.
In patient number 2, HBV-DNA was detectable by dot plot hybridization in March 1993 and September 1993 with 10 μg / 50 μl serum. 1.0 μg of HBV-DNA is 2.3 × 10 ⁶ Equivalent to the HBV genome of this patient, the serum of this patient is approximately 4.6 × 10 6 per ml. ⁷ It was thought to contain virus particles. HBsAg could be detected in the first sample but not in the second sample. In January 1994, HBV-DNA could only be detected by PCR and HBsAg was still undetectable.
Study on monoclonal antibody binding
From studies on binding in patient numbers 1 and 2 and HBsAg positive patients used as controls (adw), serum HBsAg binds to all three monoclonal antibodies in patient number 2 and the AusRIA II kit. Of polyclonal anti-HBs (Table 2).
Sequencing of PreS-S gene and PreC / C region
The S gene is composed of 687 bp in isolate 1, 690 bp in isolate 2 and 681 bp in the wild type. The S nucleotide and amino acid sequences of the mutants were compared to known sequences of the same subtype (adw) and even wild type strains from HBeAg-positive carriers from the same region (as shown in Table 3). .
Sequencing results clearly showed that there was an insert in the S gene (FIG. 4). The inserted sequence further encodes two amino acids (Arg-Ala) between the 122 and 123 codons in isolate 1 and 3 between the 123 and 124 codons in isolate 2. Is further encoded (Arg-Gly-Ala) (FIG. 5). These insertions occur immediately before the “a” determinant of HBsAg. Such insertions were not described in the reported sequences of known HBV subtypes and were missing consensus sequences from 30 Chinese HBsAg positive patients from the same region of China .
The results of direct sequencing were confirmed by cloning sequencing. Of the 10 clones from the first serum taken from patient number 2, 8 clones have in-phase insertion as described above, 2 of which are wild type, All 15 clones from the second and third serum samples were mutated. Thus, because the first serum from patient number 2 has the form of a mixture of wild-type and mutant viruses, the mutant gradually emerges and later in the serum Showed to be dominant. There were no amino acid deletions or substitutions as described above in the Pre-S1 and Pre-S2 genes.
Hydrophobic plot
When performed according to the method of Kyte and Doolittle, the mean hydrophobicity index was -0.48 for amino acids 122-137 of the wild type "a" determinant, It was 0 in the 122th to 139th amino acids of the isolate 1 and further −0.89 in the 122nd to 140th amino acids of the isolate 2. Thus, the mutant “a” determinant was more hydrophobic than the same region from the wild type virus.

Claims (16)

In the hepatitis B surface antigen protein (mHBsAg) that exhibits the antigenicity of the surface antigen of HBV, the protein is composed of arginine and alanine between the codon at position 122 and the codon at position 124 of the HBsAg sequence. Hepatitis B surface antigen protein characterized in that it consists of an envelope region in which the inserted antigenicity is modified in order or in the order of arginine, glycine and alanine. The mHBsAg according to claim 1, which is the same as the S protein of normal HBsAg except that the amino acid residue is inserted between the codon at position 122 and the codon at position 124. The mHBsAg according to claim 1 or 2, wherein a codon is inserted between the codon at position 122 and the codon at position 123. The mHBsAg according to claim 1 or 2, wherein the codon is inserted between the codon at position 123 and the codon at position 124. The mHBsAg according to any one of claims 1 to 4, wherein the mHBsAg comprises a Pre-S sequence. The mHBsAg according to any one of claims 1 to 5, which has a purity of 75% or more. An expression vector comprising a DNA sequence encoding the mHBsAg according to any one of claims 1 to 6. A host transformed with the vector according to claim 7. The method according to any one of claims 1 to 6, further comprising the step of culturing the host according to claim 8 in an appropriate culture medium and further purifying mHBsAg produced to a required purity. Preparation method of mHBsAg. A kit intended for in vitro detection for the diagnosis of anti-mHBsAg antibodies in a biological medium, characterized in that it comprises:
(A) mHBsAg according to any one of claims 1 to 6;
And (b) means used to detect the antigen-antibody reaction. An antibody preparation comprising an antibody against mHBsAg according to any one of claims 1 to 6 intended for use in diagnosis, prevention or treatment of hepatitis B infection in humans. A DNA sequence encoding the mHBsAg according to any one of claims 1 to 6. The DNA sequence according to claim 12 , which has arginine and alanine or arginine, glycine and alanine inserted in the following order between the codon at position 122 and the codon at position 124 of the mHBsAg sequence. fragment. The DNA sequence of claim 12 or the claim of claim 12 for use in an in vitro detection method for the diagnosis of mutant HBV encoding mHBsAg according to any of claims 1-6. 14. A fragment of the DNA sequence according to item 13. The DNA sequence of claim 12 or the claim of claim 12 used as a probe for determining the presence of a mutant HBV encoding the mHBsAg of any of claims 1-6. 14. A fragment of the DNA sequence according to item 13. 1) PCR amplification using the codon at position 122 of the HBsAg sequence and a primer adjacent to the codon at position 124; and 2) the amplified sequence is the codon at position 122 and position 124 of the HBsAg sequence. The method according to any one of claims 1 to 6, further comprising the step of determining whether or not the codon according to any one of claims 1 to 6 is inserted between codons at the position. A method for determining the presence of a mutated HBV encoding mHBsAg.

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